**2.1. VADS constructed with metal nanoparticles (mNPs) formed by self-aggregation**

Alum is the micron-sized aggregates of water-insoluble aluminum salts and is the first substance that was discovered able to boost the efficacy of vaccines and coined the term "vaccine adjuvant," a concept which was put forward when scientists came to realize that certain materials irrelevant to pathogens but able to enhance immunoresponse induced by vaccine [14]. Since 1926, when first being used as an adjuvant, alum, such as aluminum phosphate, aluminum potassium sulfate, and aluminum hydroxide had been the only clinically used adjuvant in many subunit vaccines as well as the inactivated pathogen-based vaccines, until the approval of adjuvant calcium phosphate in diphtheria/pertussis (DT) vaccines [15]. Subsequently, an O/W nanoemulsions formed of squalene/Span 85/Tween 80, called MF59® was marketed as a VADS for delivering an influenza vaccine (Fluad®) in 1997, followed by AS04 (MPL/alum mixture) for delivering human papillomavirus (HPV, Cervarix®) and hepatitis B virus (HBV, Fendrix®) vaccines [16]. Alum forms a micron-sized VADS by just mixing the insoluble salt with other vaccine components or Ags and tends to function eliciting humoral over cellular immunity when intramuscularly administered to humans. Though having successfully been used for nearly a century in human vaccines against numerous infectious diseases, such as hepatitis A and B, diphtheria-tetanus-pertussis (DTaP, Tdap), Haemophilus influenzae type b (Hib), HPV, and pneumococcus infection, alum is still argued to be associated with a potential risk for causing autoimmunity, long-term brain inflammation and neurological complications, as evidenced by the observation of severe disorders in recipients of alum-adjuvanted vaccines [17].

producing a particulate vaccine with a hydrodynamic size of 60 nm and found that the small core-shell assemblies induced in mice a 3-fold increase of anti-Ag titers 3 weeks post-injection, compared to a commercial aluminum phosphate adjuvant, suggesting that CaP-NPs may be an effective VADS delivery of vaccines [23]. Morcol et al. demonstrated that CaP-NPs were also a good VADS for the inactivated influenza A/CA/04/2009 (H1N1pdm) vaccine and could enormously boost production in the intramuscularly vaccinated mice of hemagglutination inhibition (HAI), virus neutralization (VN), and IgG antibody titers, at all dose levels, relative to the nonadjuvanted vaccine. In particular, the CaP-NP vaccine equally protected mice against influenza virus at 1/3 of the Ag dose of the nonadjuvanted or alum-adjuvanted vaccines, indicating that CaP-NPs are an promising VADS which may play a crucial role in production of a dose-sparing vaccine which is of a great importance during, in particularly, an influenza pandemic [24]. Also, Powell and coworkers constructed calcium carbonate NPs which had an average diameter of 200 nm and based on opposite charge attraction, coated

Vaccine Adjuvant Delivery Systems Constructed Using Biocompatible Nanoparticles Formed…

http://dx.doi.org/10.5772/intechopen.79905

with polylysine and polyglutamic acid and showed that this type of the CaCO3

**2.2. VADS constructed with emulsions formed by self-assembly of surfactants**

Emulsions are formed of two immiscible liquid phases, generally oil phase and water phase, with one phase organized into small droplets (inner phase), which, depending on composition and manufacturing process, have a size in a range of from tens of nanometers to several

cytokines, proving CaCO3

VADS could efficiently facilitate maturation of DCs, which were simultaneously induced capable of cross-presentation of Ags. Notably, after a single injection in mice, CaCO3

induced strong humoral and cellular immunity without triggering secretion of inflammatory

NPs are an efficient and safe VADS [25].

Gold nanoparticles (AuNPs) have unique physicochemical properties, such as an ultra-small size, large surface area to mass ratio, and high surface reactivity, presence of surface plasmon resonance (SPR) bands, biocompatibility and ease of surface functionalization, allowing this type of mNPs able to act as a versatile VADS bearing numerous beneficial features including, particularly, targeted delivery and stimulus-sensitive release. Chen et al. engineered gold NPs (AuNPs) with sizes ranging from 2 to 50 nm conjugated with foot-and-mouth disease virus associated peptide Ags and proved that gold NPs with a size of ranging in 2–17 nm induced strong humoral response, which was correlated to spleen uptake of gold NPs [26]. Gill's group prepared gold NPs conjugated with M2e peptide, an extracellular domain of influenza A virus ion channel membrane matrix protein 2 (M2e) and demonstrated that intranasal administration to mice of AuNP-M2e plus soluble CpG induced lung B cell activation and robust serum anti-M2e antibody response, resulting in high levels of both IgG1 and IgG2a subtypes [27]. Also, the group revealed that the antibodies generated by AuNP-M2e/CpG stimulation could bind to the homotetrameric form of M2 expressed on Madin-Darby canine kidney (MDCK) cells, which as an immunosorbent had been infected with H1N1, H3N2 or H5N1 strain of influenza viruses. Moreover, mice intranasally immunized with AuNP-M2e/CpG obtained 100, 92, and 100% protection against lethal challenges with A/California/04/2009 (H1N1pdm) pandemic strain, A/Victoria/3/75 (H3N2), and the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1), respectively, proving AuNP-M2e/CpG a promising VADS for developing a universal influenza vaccine, a desired Holy Grail for controlling the most prevalent infections [27].

NP-based

NPs

13

Thus, frustrated by the reactogenicity and the injury adverse effects associated alum while expected to enhance its capability to induce humoral and even cellular immunoresponses, researchers have for years endeavored to reshape the micron-sized salt adjuvant in mainly two ways: forging the micron-sized salt into NPs and coating surfaces with biocompatible materials. Recently, to develop an effective HIV vaccine, which is known a huge challenge almost since this virus discovery, Neutra's group conjugated peptide epitopes derived from HIV-1 gp120 glycoprotein to the Al2 O3 NPs with a size of about 350 nm, which showed able to stimulate the moderate antibody responses after intraperitoneal injection but failed to stimulate mucosal immunity [18]. Also, Cui's group engineered 112 nm-sized aluminum hydroxide NPs and aluminum oxyhydroxide nanosticks with a length of 80 nm, long aspect ratio of 10 and low degree of crystallinity and showed that both aluminum NPs were able to facilitate in vitro APC uptake of the loaded protein Ags and induced in mice a stronger Ag-specific antibody response but milder local inflammation in the injection sites, compared with traditional aluminum microparticles [19, 20]. Furthermore, aluminum NPs proved able to stimulate in vitro APCs to produce uric acid, and, when injected into peritoneal cavity of mice, induced production of increased levels of uric acid, to contrast micron alum which did not in either case. The results suggest that aluminum has a stronger adjuvant activity in the form of NPs, as opposed to microparticles, may be partially attributed to their higher ability to induce endogenous danger signals such as uric acid [21].

Based on the phosphophilicity of aluminum, Wang and coworkers engineered the phospholipid bilayer-coated aluminum nanoparticles (PLANs) formed via chemisorption between phospholipid and aluminum using a procedure of reverse ethanol injection-lyophilization (REIL) [7]. The researchers demonstrated that the anhydrous Ag-PLANs had a high stability satisfying the prerequisite requirements for distribution with the controlled temperature chain instead of the integrated cold chain [22] and that upon rehydration the Ag-carried PLANs could be instantly reconstituted to form an aqueous dispersion maintaining vaccine activity. Further exploration confirmed that the PLANs remarkably enhanced APC uptake of the delivered vaccines and when given subcutaneously to mice, induced more robust Ag-specific humoral as well as cellular immunoresponses, while stimulated less local inflammations, in comparison to microparticle alum, proving that the PLANs are an efficient VADS and possess numerous advantages over alum, which has been the widely used for clinical immunization for nearly a century [7].

In recent years, other types of NPs made of metal substances, such as calcium and gold, have also become a popular VADS, owing to their certain unique physicochemical properties including inertness with good biocompatibility, facile surface modification with functional molecules, and easy size and shape control. Chiu and coworkers coated the 25 nm-sized amorphous cores of calcium phosphate nanoparticles (CaP-NPs) with peptide Ags, thus producing a particulate vaccine with a hydrodynamic size of 60 nm and found that the small core-shell assemblies induced in mice a 3-fold increase of anti-Ag titers 3 weeks post-injection, compared to a commercial aluminum phosphate adjuvant, suggesting that CaP-NPs may be an effective VADS delivery of vaccines [23]. Morcol et al. demonstrated that CaP-NPs were also a good VADS for the inactivated influenza A/CA/04/2009 (H1N1pdm) vaccine and could enormously boost production in the intramuscularly vaccinated mice of hemagglutination inhibition (HAI), virus neutralization (VN), and IgG antibody titers, at all dose levels, relative to the nonadjuvanted vaccine. In particular, the CaP-NP vaccine equally protected mice against influenza virus at 1/3 of the Ag dose of the nonadjuvanted or alum-adjuvanted vaccines, indicating that CaP-NPs are an promising VADS which may play a crucial role in production of a dose-sparing vaccine which is of a great importance during, in particularly, an influenza pandemic [24]. Also, Powell and coworkers constructed calcium carbonate NPs which had an average diameter of 200 nm and based on opposite charge attraction, coated with polylysine and polyglutamic acid and showed that this type of the CaCO3 NP-based VADS could efficiently facilitate maturation of DCs, which were simultaneously induced capable of cross-presentation of Ags. Notably, after a single injection in mice, CaCO3 NPs induced strong humoral and cellular immunity without triggering secretion of inflammatory cytokines, proving CaCO3 NPs are an efficient and safe VADS [25].

intramuscularly administered to humans. Though having successfully been used for nearly a century in human vaccines against numerous infectious diseases, such as hepatitis A and B, diphtheria-tetanus-pertussis (DTaP, Tdap), Haemophilus influenzae type b (Hib), HPV, and pneumococcus infection, alum is still argued to be associated with a potential risk for causing autoimmunity, long-term brain inflammation and neurological complications, as evidenced by

Thus, frustrated by the reactogenicity and the injury adverse effects associated alum while expected to enhance its capability to induce humoral and even cellular immunoresponses, researchers have for years endeavored to reshape the micron-sized salt adjuvant in mainly two ways: forging the micron-sized salt into NPs and coating surfaces with biocompatible materials. Recently, to develop an effective HIV vaccine, which is known a huge challenge almost since this virus discovery, Neutra's group conjugated peptide epitopes derived from

to stimulate the moderate antibody responses after intraperitoneal injection but failed to stimulate mucosal immunity [18]. Also, Cui's group engineered 112 nm-sized aluminum hydroxide NPs and aluminum oxyhydroxide nanosticks with a length of 80 nm, long aspect ratio of 10 and low degree of crystallinity and showed that both aluminum NPs were able to facilitate in vitro APC uptake of the loaded protein Ags and induced in mice a stronger Ag-specific antibody response but milder local inflammation in the injection sites, compared with traditional aluminum microparticles [19, 20]. Furthermore, aluminum NPs proved able to stimulate in vitro APCs to produce uric acid, and, when injected into peritoneal cavity of mice, induced production of increased levels of uric acid, to contrast micron alum which did not in either case. The results suggest that aluminum has a stronger adjuvant activity in the form of NPs, as opposed to microparticles, may be partially attributed to their higher ability

Based on the phosphophilicity of aluminum, Wang and coworkers engineered the phospholipid bilayer-coated aluminum nanoparticles (PLANs) formed via chemisorption between phospholipid and aluminum using a procedure of reverse ethanol injection-lyophilization (REIL) [7]. The researchers demonstrated that the anhydrous Ag-PLANs had a high stability satisfying the prerequisite requirements for distribution with the controlled temperature chain instead of the integrated cold chain [22] and that upon rehydration the Ag-carried PLANs could be instantly reconstituted to form an aqueous dispersion maintaining vaccine activity. Further exploration confirmed that the PLANs remarkably enhanced APC uptake of the delivered vaccines and when given subcutaneously to mice, induced more robust Ag-specific humoral as well as cellular immunoresponses, while stimulated less local inflammations, in comparison to microparticle alum, proving that the PLANs are an efficient VADS and possess numerous advantages over alum, which has been the widely used for clinical immunization

In recent years, other types of NPs made of metal substances, such as calcium and gold, have also become a popular VADS, owing to their certain unique physicochemical properties including inertness with good biocompatibility, facile surface modification with functional molecules, and easy size and shape control. Chiu and coworkers coated the 25 nm-sized amorphous cores of calcium phosphate nanoparticles (CaP-NPs) with peptide Ags, thus

NPs with a size of about 350 nm, which showed able

the observation of severe disorders in recipients of alum-adjuvanted vaccines [17].

O3

to induce endogenous danger signals such as uric acid [21].

HIV-1 gp120 glycoprotein to the Al2

12 Immunization - Vaccine Adjuvant Delivery System and Strategies

for nearly a century [7].

Gold nanoparticles (AuNPs) have unique physicochemical properties, such as an ultra-small size, large surface area to mass ratio, and high surface reactivity, presence of surface plasmon resonance (SPR) bands, biocompatibility and ease of surface functionalization, allowing this type of mNPs able to act as a versatile VADS bearing numerous beneficial features including, particularly, targeted delivery and stimulus-sensitive release. Chen et al. engineered gold NPs (AuNPs) with sizes ranging from 2 to 50 nm conjugated with foot-and-mouth disease virus associated peptide Ags and proved that gold NPs with a size of ranging in 2–17 nm induced strong humoral response, which was correlated to spleen uptake of gold NPs [26]. Gill's group prepared gold NPs conjugated with M2e peptide, an extracellular domain of influenza A virus ion channel membrane matrix protein 2 (M2e) and demonstrated that intranasal administration to mice of AuNP-M2e plus soluble CpG induced lung B cell activation and robust serum anti-M2e antibody response, resulting in high levels of both IgG1 and IgG2a subtypes [27]. Also, the group revealed that the antibodies generated by AuNP-M2e/CpG stimulation could bind to the homotetrameric form of M2 expressed on Madin-Darby canine kidney (MDCK) cells, which as an immunosorbent had been infected with H1N1, H3N2 or H5N1 strain of influenza viruses. Moreover, mice intranasally immunized with AuNP-M2e/CpG obtained 100, 92, and 100% protection against lethal challenges with A/California/04/2009 (H1N1pdm) pandemic strain, A/Victoria/3/75 (H3N2), and the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1), respectively, proving AuNP-M2e/CpG a promising VADS for developing a universal influenza vaccine, a desired Holy Grail for controlling the most prevalent infections [27].

#### **2.2. VADS constructed with emulsions formed by self-assembly of surfactants**

Emulsions are formed of two immiscible liquid phases, generally oil phase and water phase, with one phase organized into small droplets (inner phase), which, depending on composition and manufacturing process, have a size in a range of from tens of nanometers to several microns, and are dispersed in a distinct continuous phase (outer phase) under stabilization by an interfacial surfactant layer. Emulsions, based on structural characteristics, are made of three classical types of single emulsions, double emulsions and Pickering emulsions: single emulsions include oil-in-water (O/W) type denoting oil droplets being emulsified in a bulk aqueous phase, and vice versa, the water-in-oil (W/O) type; double emulsions include O/W/O and W/O/W emulsions; and Pickering emulsions are a special type with an emulsifier of solid NPs replacing surfactants [28].

anti-Ag immunity. MF59 became the first emulsion-based VADS approved for delivering the seasonal influenza vaccine of Fluad® for human immunization in 1997 and followed by AS03, which is a 200 nm-sized O/W emulsion consisting of squalene/DL-a-tocopherol/Tween 80 and was approved for human use in GSK's A/H1N1 pandemic flu vaccine Pandemrix® [33]; and then AF03, which is a 80 nm-sized O/W emulsion consisting of squalene/polyoxyethylene cetyl-stearyl ether/sorbitan oleate/mannitol and was approved for clinical immunization in

Vaccine Adjuvant Delivery Systems Constructed Using Biocompatible Nanoparticles Formed…

http://dx.doi.org/10.5772/intechopen.79905

15

Now, novel types of emulsions are still actively formulated using various functional materials to constitute a VADS possessing desired properties, including high potent immunogenicity, targeting delivery of vaccines toward draining lymph nodes (dLNs) and APCs, enhanced cellular uptake, controlled release of Ags, rendering vaccine lysosome escape, and directing immunoresponses toward the Th1/Th2 type biased or balanced pathway [32]. Meanwhile, attempts in pushing into clinical trials of emulsion VADSs for delivery of cancer vaccines and other applications have also increasingly continued and are accompanied by endeavors in shedding light on the mechanisms involved in the action of emulsion adjuvants. Recently, Schmidt et al. using squalane as an O and distearoylphosphoethanolamine (DSPE) as an emulsifier engineered the TLR3a poly(I:C)-entrapping cationic nanoemulsions with a size of 200 nm and demonstrated that when given to mice the cationic nanoemulsions drained rapidly to the LNs and activated cross-presenting DCs, MPs as well as B cells, resulting in strong Ag-specific CD8+ T-cell responses [35]. The results suggest the squalane-based cationic nanoemulsions may be a promising VADS with the ability to induce strong CTL responses, offering an alternative way to make vaccines against pathogens that can hardly be protected without activated CTLs. Interestingly, using squalene as O but the 100 nm-sized poly(D, L-lactic-*co*-glycolide) (PLGA) NPs as a stabilizer, Ma and coworkers formulated 2 μm-sized Pickering emulsions as a VADS, which retained the force-dependent deformability and lateral mobility of loaded Ags [36]. Mouse experiments proved that the Pickering emulsions enhanced the recruitment, Ag uptake, and activation of APCs which initiated robust humoral as well as cellular immunoresponses, which effectively supported mice to survive a lethal challenge of influenza virus. The outcomes hint that the pliability of vaccine carriers and lateral mobility of Ags may well count in triggering immune reactions and, as such, may well

In summary, as one of a few types of VADSs that have been approved for human use, certain types of emulsions prove by numerous clinical and preclinical evaluations capable of eliciting strong humoral and/or cellular immunity against heterologous pathogens meanwhile maintain an excellent safety profile, depending on the components as well as the structural characteristics of this fluid carrier. Further development of emulsion VADSs may focus on elucidating the mechanisms underlying the immunopotentiating functions in regard of particularly the relationship between emulsion efficacy, systematic characteristics, and molecular structure of squalene, squalane or other unidentified active materials [32]. Further efforts may well be committed to improving the stability of emulsions to construct a VADS allowing the products to be distributed, at least for some time, out of the cold chain [37], thus facilitating global vaccination against various infections in, especially, some low-income countries or

Sanofi Pasteur's pandemic influenza vaccine, Humenza® [34].

be taken into account when developing certain types of VADS.

districts, where integral cold chain may not be available.

Notably, the emulsions formed of special oils, such as lanolin oil, cottonseed oil, and paraffin oil, were found, like alum, in some serendipitous way, of adjuvanticity in the early twentieth century and have ever since been widely used as a VADS to produce vaccines against pathogens. For example, lipovaccines used in the 1920s were in fact the formulations consisting of killed bacterial vaccines suspended in lanolin or cottonseed oils and proved able to induce immunoresponses with additional functions of dose spare and stability enhancement [29]. Freund adjuvants are the mostly known potent emulsion-based VADS including two types: incomplete Freund adjuvant(IFA), which is essentially a viscous crude W/O emulsion containing Ags in water phase using mineral paraffin as an oil phase and mannide monooleate as a surfactant; complete Freund adjuvant (CFA), which forms by addition to IFA of heat-killed mycobacteria (*Mycobacterium tuberculosis*) and has thus a high immunostimulating potency but also a high reactogenic toxicity, rendering the adjuvant to be used only in veterinary vaccines [30]. Though IFA is rather safe compared to CFA and was actually administered to hundreds of thousands of humans as an adjuvant in polio and influenza vaccines in the mid-twentieth century, the severe local reactogenicity excluded the adjuvant from continuing clinical use [31].

Discarding the flaws of unacceptable toxicity and uncertain component associated with early emulsion adjuvants, modern emulsions as a VADS are usually formulated with well-defined factors, such as particle size, component and concentrations, and compatibility with antigens as well as human bodies, which are related to efficacy, safety, and stability [32]. Important lessons highlighted by early emulsion vaccines and deep insights into problems arising in use of the adjuvant inspired researchers to commit to developing an emulsion VADS with clear thoughts in several issues: (1) using biodegradable oil and the surfactants with an established safety profile in humans; (2) using the O/W instead of W/O emulsions to lower oil content for enhancing tolerability as well as the ease of use due to reduced viscosity; (3) enhancing potency with emulsions having a size <500 nm to promote APC uptake. As a result of the efforts directed toward these aspects, a breakthrough was made in the development of emulsion VADS in the 1980s when the squalene was explored as the oil phase of emulsions, which were thus rendered with an acceptable reactogenicity profile and potent adjuvant effects and were subsequently licensed as several proprietary products, including MF59 by Novartis, AS03® by GSK and AF03 by Sanofi Pasteur. MF59 is an O/W emulsion which is produced with a microfluidizer (MF) and contains squalene oil droplets stabilized by surfactants Tween 80 and Span85 guaranteeing the size of 160 nm for sterilization by filtration and as a VADS has proven of potent immunogenicity and low reactogenicity for a range of Ags [16]. MF59 can induce robust immunoresponses through triggering vaccinated tissue-resident immune cells to secrete a number of chemokines, which recruit other immunocytes to amplify the chemokine gradient, resulting in a significant signal magnification and immune cell influx to establish anti-Ag immunity. MF59 became the first emulsion-based VADS approved for delivering the seasonal influenza vaccine of Fluad® for human immunization in 1997 and followed by AS03, which is a 200 nm-sized O/W emulsion consisting of squalene/DL-a-tocopherol/Tween 80 and was approved for human use in GSK's A/H1N1 pandemic flu vaccine Pandemrix® [33]; and then AF03, which is a 80 nm-sized O/W emulsion consisting of squalene/polyoxyethylene cetyl-stearyl ether/sorbitan oleate/mannitol and was approved for clinical immunization in Sanofi Pasteur's pandemic influenza vaccine, Humenza® [34].

microns, and are dispersed in a distinct continuous phase (outer phase) under stabilization by an interfacial surfactant layer. Emulsions, based on structural characteristics, are made of three classical types of single emulsions, double emulsions and Pickering emulsions: single emulsions include oil-in-water (O/W) type denoting oil droplets being emulsified in a bulk aqueous phase, and vice versa, the water-in-oil (W/O) type; double emulsions include O/W/O and W/O/W emulsions; and Pickering emulsions are a special type with an emulsifier of solid

Notably, the emulsions formed of special oils, such as lanolin oil, cottonseed oil, and paraffin oil, were found, like alum, in some serendipitous way, of adjuvanticity in the early twentieth century and have ever since been widely used as a VADS to produce vaccines against pathogens. For example, lipovaccines used in the 1920s were in fact the formulations consisting of killed bacterial vaccines suspended in lanolin or cottonseed oils and proved able to induce immunoresponses with additional functions of dose spare and stability enhancement [29]. Freund adjuvants are the mostly known potent emulsion-based VADS including two types: incomplete Freund adjuvant(IFA), which is essentially a viscous crude W/O emulsion containing Ags in water phase using mineral paraffin as an oil phase and mannide monooleate as a surfactant; complete Freund adjuvant (CFA), which forms by addition to IFA of heat-killed mycobacteria (*Mycobacterium tuberculosis*) and has thus a high immunostimulating potency but also a high reactogenic toxicity, rendering the adjuvant to be used only in veterinary vaccines [30]. Though IFA is rather safe compared to CFA and was actually administered to hundreds of thousands of humans as an adjuvant in polio and influenza vaccines in the mid-twentieth century, the severe local reactogenicity excluded the adjuvant from continuing clinical use [31].

Discarding the flaws of unacceptable toxicity and uncertain component associated with early emulsion adjuvants, modern emulsions as a VADS are usually formulated with well-defined factors, such as particle size, component and concentrations, and compatibility with antigens as well as human bodies, which are related to efficacy, safety, and stability [32]. Important lessons highlighted by early emulsion vaccines and deep insights into problems arising in use of the adjuvant inspired researchers to commit to developing an emulsion VADS with clear thoughts in several issues: (1) using biodegradable oil and the surfactants with an established safety profile in humans; (2) using the O/W instead of W/O emulsions to lower oil content for enhancing tolerability as well as the ease of use due to reduced viscosity; (3) enhancing potency with emulsions having a size <500 nm to promote APC uptake. As a result of the efforts directed toward these aspects, a breakthrough was made in the development of emulsion VADS in the 1980s when the squalene was explored as the oil phase of emulsions, which were thus rendered with an acceptable reactogenicity profile and potent adjuvant effects and were subsequently licensed as several proprietary products, including MF59 by Novartis, AS03® by GSK and AF03 by Sanofi Pasteur. MF59 is an O/W emulsion which is produced with a microfluidizer (MF) and contains squalene oil droplets stabilized by surfactants Tween 80 and Span85 guaranteeing the size of 160 nm for sterilization by filtration and as a VADS has proven of potent immunogenicity and low reactogenicity for a range of Ags [16]. MF59 can induce robust immunoresponses through triggering vaccinated tissue-resident immune cells to secrete a number of chemokines, which recruit other immunocytes to amplify the chemokine gradient, resulting in a significant signal magnification and immune cell influx to establish

NPs replacing surfactants [28].

14 Immunization - Vaccine Adjuvant Delivery System and Strategies

Now, novel types of emulsions are still actively formulated using various functional materials to constitute a VADS possessing desired properties, including high potent immunogenicity, targeting delivery of vaccines toward draining lymph nodes (dLNs) and APCs, enhanced cellular uptake, controlled release of Ags, rendering vaccine lysosome escape, and directing immunoresponses toward the Th1/Th2 type biased or balanced pathway [32]. Meanwhile, attempts in pushing into clinical trials of emulsion VADSs for delivery of cancer vaccines and other applications have also increasingly continued and are accompanied by endeavors in shedding light on the mechanisms involved in the action of emulsion adjuvants. Recently, Schmidt et al. using squalane as an O and distearoylphosphoethanolamine (DSPE) as an emulsifier engineered the TLR3a poly(I:C)-entrapping cationic nanoemulsions with a size of 200 nm and demonstrated that when given to mice the cationic nanoemulsions drained rapidly to the LNs and activated cross-presenting DCs, MPs as well as B cells, resulting in strong Ag-specific CD8+ T-cell responses [35]. The results suggest the squalane-based cationic nanoemulsions may be a promising VADS with the ability to induce strong CTL responses, offering an alternative way to make vaccines against pathogens that can hardly be protected without activated CTLs. Interestingly, using squalene as O but the 100 nm-sized poly(D, L-lactic-*co*-glycolide) (PLGA) NPs as a stabilizer, Ma and coworkers formulated 2 μm-sized Pickering emulsions as a VADS, which retained the force-dependent deformability and lateral mobility of loaded Ags [36]. Mouse experiments proved that the Pickering emulsions enhanced the recruitment, Ag uptake, and activation of APCs which initiated robust humoral as well as cellular immunoresponses, which effectively supported mice to survive a lethal challenge of influenza virus. The outcomes hint that the pliability of vaccine carriers and lateral mobility of Ags may well count in triggering immune reactions and, as such, may well be taken into account when developing certain types of VADS.

In summary, as one of a few types of VADSs that have been approved for human use, certain types of emulsions prove by numerous clinical and preclinical evaluations capable of eliciting strong humoral and/or cellular immunity against heterologous pathogens meanwhile maintain an excellent safety profile, depending on the components as well as the structural characteristics of this fluid carrier. Further development of emulsion VADSs may focus on elucidating the mechanisms underlying the immunopotentiating functions in regard of particularly the relationship between emulsion efficacy, systematic characteristics, and molecular structure of squalene, squalane or other unidentified active materials [32]. Further efforts may well be committed to improving the stability of emulsions to construct a VADS allowing the products to be distributed, at least for some time, out of the cold chain [37], thus facilitating global vaccination against various infections in, especially, some low-income countries or districts, where integral cold chain may not be available.
